Helicobacter pylori is a bacterial pathogen that chronically colonizes the stomach of half of the human population worldwide. Infection by H. pylori can lead to the development of gastric pathologies such as ulcers and adenocarcinoma, which causes up to 800,000 deaths in the world each year. Persistent colonization by H. pylori relies on regulation of the expression of adaptation-related genes. One major level of such control is posttranscriptional regulation, which, in H. pylori, largely relies on a multiprotein molecular machine, an RNA degradosome, that we previously discovered. In this study, we established that the two protein partners of this machine are associated with the membrane of H. pylori. Using cutting-edge microscopy, we showed that these complexes assemble into hubs whose formation is regulated by free RNA and scaled with bacterial size and growth phase. Organelleless cellular compartmentalization of molecular machines into hubs emerges as an important regulatory level in bacteria. Helicobacter pylori is composed of the essential ribonuclease RNase J and RhpA, its sole DEAD box RNA helicase, and plays a major role in the regulation of mRNA decay and adaptation to gastric colonization. Here, the subcellular localization of the H. pylori RNA degradosome was investigated using cellular fractionation and both confocal and superresolution microscopy. We established that RNase J and RhpA are peripheral inner membrane proteins and that this association was mediated neither by ribosomes nor by RNA nor by the RNase Y membrane protein. In live H. pylori cells, we observed that fluorescent RNase J and RhpA protein fusions assemble into nonpolar foci. We identified factors that regulate the formation of these foci without affecting the degradosome membrane association. Flotillin, a bacterial membrane scaffolding protein, and free RNA promote focus formation in H. pylori. Finally, RNase J-GFP (RNase J-green fluorescent protein) molecules and foci in cells were quantified by three-dimensional (3D) single-molecule fluorescence localization microscopy. The number and size of the RNase J foci were found to be scaled with growth phase and cell volume as previously reported for eukaryotic ribonucleoprotein granules. In conclusion, we propose that membrane compartmentalization and the regulated clustering of RNase J-based degradosome hubs represent important levels of control of their activity and specificity.Posttranscriptional regulation is a major level of gene expression control in any cell. In bacteria, multiprotein machines called RNA degradosomes are central for RNA processing and degradation, and some were reported to be compartmentalized inside these organelleless cells. The minimal RNA degradosome of the important gastric pathogen  Escherichia coli, Caulobacter crescentus, or Mycobacterium tuberculosis  are key enzymes in posttranscriptional regulation, involved in RNA maturation and degradation. RNases often act in multiprotein complexes that are designated exosomes in Eukarya and Archaea and RNA degradosomes in bacteria and chloroplasts  and a reduced number of transcriptional regulators. Therefore, posttranscriptional regulation has been proposed to play a major role in the adaptive response of H. pylori  are increased more than 4-fold in the mutant in comparison with the parental wild-type strain  fluorescent fusion proteins form short-lived foci and rapidly diffuse across the inner membrane  lacks a membrane anchor and is cytoplasmic  fusion was observed to have a homogeneous membrane localization  Prediction of RNase J transmembrane domains by the TMHMM, PureseqTM, and TMPred algorithms and of intrinsically disordered regions (IDRs) by the IUPred2A algorithm. (D) Prediction of RhpA transmembrane and intrinsically disordered regions performed as described for panel C. (E) Prediction of RNase Y transmembrane regions performed as described for panel C. Download FIG S1, TIF file, 2.3 MB.(A) Western blots with antibodies against AmiE (soluble extract fraction protein), MotB (inner membrane fraction protein), and BabA (outer membrane fraction protein) on samples subjected to cellular fractionation. (B) Western blots with antibodies against RNase J and RhpA of Copyright © 2020 Tejada-Arranz et al.2020Tejada-Arranz et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2CO3; pH 11); and (iii) a high salt concentration (2 M NaCl). The three treatments consistently dissociated both RhpA and RNase J from the pelleted membrane fraction  or CFP (right) alone from plasmid pILL2157. Fluorescence is distributed all over the cell. (E) Immunofluorescence confocal microscopy of fixed H. pylori cells expressing RNase J-FLAG (left) and RhpA-FLAG (right) with primary antibodies against the FLAG-tag and secondary anti-mouse AF488 antibody. (F) The RNase J-GFP foci remain static inside the cells. The displacement of foci was analyzed during 175 min either in live bacteria (n = 285 foci) or in fixed bacteria (n = 696 foci) as a control. The small change measured in the live bacteria is below the pixel area and thus nonsignificant. MSD, mean square displacement. Download FIG S2, PDF file, 1.0 MB.(A) Schematic representation of the genetic organization of the chromosomal regions of strain B128 expressing L9-V5, RNase J-GFP, RhpA-CFP, RNase J-FLAG, and RhpA-FLAG in the corresponding strains. KanaR, kanamycin resistance cassette; ApraR, apramycin resistance cassette; CmR, chloramphenicol resistance cassette. (B) Representative phase-contrast microscopy images of the B128 wild type and strains expressing RNase J-GFP or RhpA-CFP. No difference in morphology is observed between these strains. (C) Western blots with antibodies against RNase J and RhpA on samples subjected to cellular fractionation of strains expressing or not expressing RNase J-GFP or RhpA-CFP. TE, total extract fraction; SE, soluble extract fraction; IM, inner membrane fraction. (D) Representative confocal fluorescence microscopy images of Copyright © 2020 Tejada-Arranz et al.2020Tejada-Arranz et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the B. subtilis, RNase J1 was reported to be associated with the membrane-bound RNase Y. However, the main localization of B. subtilis RNase J1 seems to be cytoplasmic  is located immediately downstream of the rhpA gene . The rnj-gfp and rhpA-cfp fusion genes were introduced into the chromosome and expressed from their native promoters, replacing the original copies of rnj and rhpA, respectively  static and (ii) nonpolar but rather randomly distributed along the cell.Our data show that the proteins of the As we showed that the RNase J-GFP fusion protein localizes like the native RNase J, we decided to use this fusion to further investigate what factors might influence focus formation. First, we quantified the number of foci per cell by automatically detecting the focus particles and assigning them to the nearest nucleoid. Every measure was normalized to the area of each nucleoid in order to avoid an overestimation of the number of foci due to dividing cells. The median number of foci per nucleoid was about 3 during the exponential-growth phase, with a range between 0 and 6 .H. pylori bacteria in stationary phase, all cells had a spiral shape. We observed that the median number of detected RNase J-GFP foci steadily reduced throughout the growth phase, with medians of 2.4 foci/cell at 20 h , 0.95 foci/cell at 36 h, and 0 foci/cell at 43 h (stationary-phase cells)  . Cell fre cells) .C. crescentus relies on free mRNAs  than with chloramphenicol (36% reduction)  in ee mRNAs . Therefoduction)  comparedduction) , an antiFurthermore, we examined whether the membrane localization of RNase J was perturbed by either the growth phase or the antibiotic treatments and we found that RNase J remained associated with the inner membrane .10.1128/mBio.01173-20.3FIG S3rny, ΔfloA, or ΔrhpA mutants. (A) Western blot with antibodies against RNase J on samples subjected to fractionation at different time points along the growth curve. (B) Western blot with antibodies against RNase J on samples subjected to rifampicin (Rif), chloramphenicol (Cam), or puromycin (Puro) treatment and fractionation. (C) Western blot with antibodies against RNase J on samples from different mutants in exponential phase that were subjected to fractionation. TE, total extract fraction; SE, soluble extract fraction; IM, inner membrane fraction. Download FIG S3, TIF file, 1.5 MB.The cellular distribution of RNase J-GFP is not affected during growth or upon antibiotic treatment or in ΔCopyright © 2020 Tejada-Arranz et al.2020Tejada-Arranz et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the H. pylori and that the formation of foci is not solely correlated with the amount of RNase J-GFP.These data suggest that ribosome-free mRNAs are among the factors promoting focus formation in H. pylori. However, we wondered whether these proteins might impact the formation of foci. Therefore, Δrny, ΔfloA and ΔrhpA mutations were introduced into the strain expressing RNase J-GFP and the number of foci per cell was quantified in exponential phase. We verified that the membrane localization of the RNase J-GFP fusion was not modified in these mutants , with the effect being much stronger in the flotillin-deficient mutant (We found that the median number of foci was significantly increased (by 22%) in the Δy strain . In botht mutant . Taken tH. pylori in both the exponential and stationary phases, for which we applied dSTORM , a powerful tool that allows determination of the location of individual molecules in the x, y, and z axes with 15-nm precision. For this analysis, H. pylori was fixed and permeabilized, membranes were labeled with wheat germ agglutinin (WGA) lectin coupled to Alexa Fluor 555 (WGA-AF555), and GFP was labeled with anti-GFP nanobodies coupled to sulfocyanin 5 (GFP-Cy5), allowing us to analyze the localization of RNase J-GFP at the single-molecule level.We decided to explore in greater detail the properties of the RNase J degradosome foci in 3 , irrespective of the total amount of RNase J-GFP . Thus, tH. pylori RNA degradosome. RNase J and RhpA, the two protein partners of this minimal degradosome, present no predictable membrane targeting sequences; they interact with the inner membrane through hydrophobic and electrostatic interactions and are thus peripheral membrane proteins of H. pylori. This membrane localization of the RNA degradosome is independent of RNA and relies neither on the membrane-bound RNase Y nor on the sole flotillin of H. pylori. The association of the degradosome partners with the H. pylori membrane might, however, be mediated by another protein, yet to be identified. During our previous studies, RNase J and RhpA were detected in association with purified ribosomes (70S particles and polysomes)  . The preE. coli, the degradosome is targeted to the inner membrane through an amphipathic helix of RNase E, and deletion of this membrane anchor results in a global slowdown of RNA degradation . The number of RNase J foci per cell was slightly diminished in the absence of its RhpA partner. In the absence of RNase Y, the number of foci per cell increased by 22%. One plausible explanation, supported by the antibiotic treatment data, is that focus formation depends on the amount of free RNA and would, in the mutant deficient in RNase Y, be promoted by the accumulation of its RNA targets. In the absence of the sole flotillin of H. pylori, the number of foci per cell was reduced by 46%, showing that this membrane scaffolding protein promotes focus formation. In S. aureus, FloA affects the function and oligomeric state of the membrane-bound RNase Y . This suggests that free/untranslated RNA is a factor promoting RNase J focus formation and that the number of degradosome foci is probably adjusted to the amount of free cellular RNAs, their proposed targets. Treatment with puromycin, an antibiotic whose presence leads to the dissociation of the ribosomal subunits, thereby increasing the amounts of untranslated free mRNAs, did not significantly change the number of foci per cell. Our interpretation is that, under this condition, the changes in the free RNA concentration are not sufficient to significantly impact focus formation. Altogether, our results show that RNA does not determine the membrane localization of RNase J and instead suggest that free/untranslated RNA is a factor promoting RNase J focus formation. We interpret this as an indication that these clusters might represent the active form of the RNA degradosome. In addition, we also observed that not all RNase J proteins present at the membrane were embedded in foci, an aspect that we were able to tackle with superresolution microscopy.We examined how the exposure of B. subtilis, rifampicin treatment does not change RNase Y membrane localization, although it modifies the number of RNase Y foci, and results in complete delocalization of RNase J1 and J2  single-particle tracking and superresolution microscopy, it was observed previously that the number of confined/clustered RNase E molecules decreases upon rifampicin treatment  and for unrelated RNases (RNase J and RNase E), pointing to evolutionarily conserved features.In 1 and J2 , 27. In iffusion . In C. creatment . Anotherreatment . Thus, uH. pylori growth phase, resulting in almost no detectable foci in stationary phase. However, in stationary phase, the total amount of RNase J-GFP proteins was slightly diminished whereas its membrane localization was maintained. Using dSTORM, we found the number of RNase J molecules per cell in stationary phase to be approximately half that in exponential phase, and we could detect more foci than with confocal microscopy, with median numbers of 4 and 2 foci per cell in the exponential and stationary phases, respectively. Between the exponential and stationary phases, the median volume of the foci was decreased by 9-fold and contained half the number of RNase J molecules. Thus, the volume of the foci was not linearly proportional to the number of RNase J molecules that each contained, suggesting that the composition and, possibly, the activity of foci change during growth. Importantly, the mean proportion of RNase J molecules contained by the foci remained the same (about 40%) between the exponential and stationary phases.Our data discussed so far suggest that the formation of RNase J foci is not constitutive. By confocal microscopy, the number of foci was found to diminish during the H. pylori as a function of growth, and we found, similarly to what was established in E. coli, that the volume was approximately two times lower in stationary phase. We then calculated that the RNase J “cellular concentration” and the number of foci per cell were approximately constant along the growth curve and conclude that the RNase J foci were undergoing a process of cell size scaling. This is particularly interesting since eukaryotic ribonucleoprotein granules depend on cell size for assembly  are necessary only for the initial step of decay of the transcripts that are degraded in stationary phase  granules (like p-bodies or stress granules). Despite their differences, the three types of clustered bacterial degradosomes reported so far (including that of H. pylori) present striking similarities to these eukaryotic structures  that can act as intermolecular interaction hot spots. The C-terminal domain of EcoRNase E is an IDR  of RhpA ; ths an IDR . Using ts an IDR , such a  of RhpA . These pC. crescentus suggest that they are active structures . H. pylori strains were grown on blood agar base 2 (Oxoid) plates supplemented with 10% defibrinated horse blood and with the following antibiotic-antifungal cocktail: amphotericin B 2.5 μg ⋅ ml−1, polymyxin B 0.31 μg ⋅ ml−1, trimethoprim 6.25 μg ⋅ ml−1, and vancomycin 12.5 μg ⋅ ml−1. Selection of H. pylori mutants was performed using kanamycin 20 μg ⋅ ml−1, apramycin 10 μg ⋅ ml−1, or chloramphenicol 10 μg ⋅ ml−1. For liquid cultures, we used Brucella broth supplemented with 10% fetal calf serum (FCS) (Eurobio), the antibiotic-antifungal cocktail, and the selective antibiotic when necessary. H. pylori cells were grown at 37°C under microaerophilic atmosphere conditions  using an Anoxomat (Mart Microbiology) atmosphere generator.The is study  were deris study , 43. Plais study  used to 10.1128/mBio.01173-20.4TABLE S1Table S1, DOCX file, 0.03 MB.Strains, plasmids, and oligonucleotides used in this study. Download Copyright © 2020 Tejada-Arranz et al.2020Tejada-Arranz et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the H. pylori genomic DNA extractions, respectively. PCR was performed either with DreamTaq DNA polymerase (Thermo Fisher) or with Q5 High-Fidelity DNA polymerase (NEB) when the product required high-fidelity polymerase.Molecular biology experiments were performed according to standard procedures  and the H. pylori mutants were obtained by natural transformation -inducible promoter. These constructs were transformed into H. pylori and verified by PCR and sequencing and served as controls for microscopy analysis.Chromosomal deletion of the entire genes encoding RNase Y (HPB128_186g30), RhpA (HPB128_21g22), and flotillin (HPB128_21g23) was performed in strain B128. Briefly, fragments of about 500 bp upstream and downstream of the target gene were amplified by PCR (oligonucleotides are listed in eviously ) with th gfpmut2  and scfp. pylori  between 600) were concentrated or diluted to an OD600 of 2 and, when necessary, stained. The cultures were labeled with NucBlue live-cell stain ReadyProbes reagent  for DNA per the recommendations of the supplier and with 4 μg/ml FM4-64 (Thermo Fisher Scientific) for membranes. For strains carrying the RhpA-CFP fusion, only FM4-64 was used. After 20 min of incubation in the dark, bacterial samples were spotted on microscope slides with a thin layer of 1% agarose–phosphate-buffered saline (PBS). The agarose pad was covered by a coverslip, and samples were directly observed at room temperature. Images of strains expressing RNase J-GFPmut2 or RhpA-SCFP from the chromosome or GFPmut2 and SCFP from pILL2157 were obtained using a Leica confocal SP8 inverted microscope through a numerical-aperture (NA) 1.4 oil immersion 63× lens objective.Bacterial samples taken from cultures with different optical densities at 600 nm (ODThe detection was performed by the use of a photomultiplier tube (PMT) detector for the NucBlue channel and HyD for the other fluorophores. Images were analyzed using Fiji . Picturel-lysine-covered coverslips. The samples were quenched for 30 min at room temperature with 100 mM glycine and were then blocked with 5% bovine serum albumin (BSA)–PBS for 1h at room temperature. They were then incubated with anti-FLAG antibody (Sigma) (1:200)–2% BSA–PBS, washed three times with 2% BSA–PBS, incubated with anti-mouse AF488 secondary antibody (Bethyl) (1:200), washed six times with 2% BSA–PBS, and mounted on Fluoromount-G (Thermo Fisher Scientific). No signal was seen when the protocol was carried out on a strain lacking a FLAG tag. Imaging and analysis were performed as described above.The strains expressing RNase J-FLAG and RhpA-FLAG were fixed and permeabilized with 100% methanol at −20°C for 5 min. Subsequently, the bacteria were washed with PBS and deposited on poly-H. pylori, images of cells were first segmented manually using the nucleoid signal, which was homogeneous. Only those cells that could be segmented with certainty were taken. RNase J-GFP foci were detected using the blob detector function with scale space (variable target blob size) from scikit-image (To quantify the RNase J-GFP foci in it-image . The parxy plane) due to chromatic aberrations, we used a standard approach utilizing fiducial markers. Multicolor fluorescent beads  (0.2-μm-diameter microspheres) were dispersed in buffer, immobilized on a glass coverslip, and imaged in the DAPI  and GFP channels. Beads were detected in the images, and their xy positions were found with subpixel resolution using the FIJI plugin TrackMate  using the skeletonization function in the Python skimage library and then assigned the values 0 and 1 to either end of the spine (poles). For each RNase J focus, the closest point on the spine of its host nucleoid was found. The length of the spine between this point and the closest pole was divided by the total length of the spine to find the relative position (RP) of the RNase J focus along the spine. In this way, if for an RNase J focus the RP value is 0 or 1, it is then located at one of the poles, whereas if the RP is 0.5, it is then located at the middle of the spine. Finally, the frequencies of foci with RP = 0 or 1 were normalized by the perimeter of the pole tip to compensate for the accumulation of foci due to their rotational freedom around the tip. The code data are available at the following repository: To quantitatively compare the mobilities of the foci in live and fixed cells, we used mean square displacement (MSD) data as metrics. For this, foci were first tracked with subpixel resolution using the TrackMate plugin in FIJI . The resH. pylori cells were grown to an appropriate OD600  and then harvested by centrifugation and washed with PBS prior to being resuspended . Then, bacteria were disrupted by sonication in a lysis buffer containing 10 mM Tris-HCl (pH 7.4) (buffer A) and Complete protease inhibitor cocktail (Roche). Cell debris was removed by centrifugation at 16,000 × g at 4°C for 10 min, and supernatants were collected as total extracts. The supernatants were transferred to 1.5-ml ultracentrifugation tubes  and then centrifuged 45 min at 100,000 × g at 4°C in a TLA-100 ultracentrifuge (Beckman Coulter). The supernatant contained the soluble fraction, and the pellet corresponded to the total membranes. The pellet was washed once with buffer A and then resuspended in 10 mM Tris-HCl (pH 7.5)–0.1% N-lauroyl-sarcosin (Sigma-Aldrich)–Complete protease inhibitor cocktail (buffer B). After another ultracentrifugation performed under the same conditions, the supernatant contained the inner membrane and the pellet the outer membrane, which was resuspended in 10 mM Tris-HCl (pH 7.5)–1% N-lauroyl-sarcosin–Complete protease inhibitor cocktail (buffer C). At each step, each sample was suspended in the same volume to obtain samples from the same number of bacteria.The cellular fractionation protocol was adapted from a previous study . H. pyloH. pylori, we tested different treatments as described previously  or to a high salt concentration (NaCl 2 M). After ultracentrifugation for 45 min at 100,000 × g, peripheral proteins were found in the supernatant whereas integral membrane proteins were found in the pellet. Finally, treatments with 1 μg/ml RNase A (Thermo Fisher) or 20 mM EDTA (Sigma-Aldrich) were performed by adding them to the cells just after sonication, and those conditions were maintained during the first ultracentrifugation.To determine the nature of the interaction of RhpA and RNase J with the inner membrane of eviously . PeripheTo examine the effect of antibiotics on the subcellular localization of RNase J and RhpA and the RNase J-GFP focus formation, rifampicin, chloramphenicol, or puromycin was added at 100 μg/ml, 200 μg/ml, or 150 μg/ml, respectively, to cultures in late exponential phase, as described previously . CultureH. pylori RhpA, RNase J, AmiE, MotB, and BabA proteins were detected with rabbit polyclonal antibodies anti-RhpA, anti-RNase J (Proteins were loaded and separated on a 4% to 20% Mini-Protean TGX stain-free precast protein gel (Bio-Rad) and subsequently electrotransferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad) with a TransBlot Turbo system (Bio-Rad). The -RNase J , anti-Am-RNase J , anti-Mo-RNase J  at the rBacterial suspensions of the RNase J-GFP-expressing strain in exponential-growth phase (after 18 h of culture) or in stationary phase (after 40 h of culture) were fixed in 1% paraformaldehyde (Sigma)–PBS for 5 min at room temperature. Following three washes in PBS, bacteria were permeabilized using 0.05% Triton X-100–PBS. Anti-GFP nanobodies coupled with Cy5  were then added (1:250) to the bacterial suspensions and incubated for 1 h at 37°C. The cells were then washed and treated with 2 μg/ml WGA-AF555 (Thermo Fisher Scientific)–PBS. In order to (i) immobilize the bacterial cells onto the coverslips  and (ii) induce the photoswitching of Cy5, the bacteria were seeded onto STORM buffer-based pads .2D and 3D images were taken using an inverted bright-field Olympus IX83 microscope equipped with an oil immersion 100× lens objective with a high numerical aperture value (1.49). To perform fluorescence nanoscopy experiments, a SAFe360 module  was added to the camera port of the microscope. This detection module couples single-molecule localization microscopy (SMLM), supercritical angle fluorescence (SAF), and astigmatism  in a duaH. pylori cells was calculated using the dSTORM images on the basis of the xyz coordinates of the detections of the membrane marker (WGA-AF555). Clusters of detections belonging to a bacterium were identified with the XY coordinates, and the clusters were refined by filtering by the number of points in a cluster and the radius of gyration. Each cluster was converted into a 2D polygon using the Python alphashape library, and the 2D area (A) of the polygon was calculated from the xy coordinates of the underlying cluster. Using this area, the volume (V) was calculated using the expression V = H*A, where H is the height of the bacteria. The height was estimated from the span of coordinates in the z dimension of the clusters (600 nm). The volume of the bacteria might be slightly overestimated due to the localization errors in imaging; however, this should not influence the ratio of exponential-growth-phase cells to stationary-growth-phase cells.The volume of P values lower than 0.05 were considered significant. The differences between the median values derived from superresolution microscopy were analyzed with the Mann-Whitney test, and P values lower than 0.05 were considered significant.To compare the numbers of degradosome foci per cell between the different conditions, we used the nonparametric Mood’s median test, and 10.1128/mBio.01173-20.7VIDEO S3Video S3, MOV file, 2.2 MB.3D visualization of superresolution imaging data corresponding to an example of representative bacteria in exponential growth phase. The yellow-red gradient is the membrane, labeled with WGA-AF555, according to the distance to the coverslip, and the blue-green gradient is RNase J-GFP, labeled with anti-GFP-Cy5 nanobodies, according to the distance to the coverslip. Download Copyright © 2020 Tejada-Arranz et al.2020Tejada-Arranz et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01173-20.8VIDEO S4Video S4, MOV file, 2.2 MB.3D visualization of superresolution imaging data corresponding to a second example of representative bacteria in exponential growth phase. The yellow-red gradient is the membrane, labeled with WGA-AF555, according to the distance to the coverslip, and the blue-green gradient is RNase J-GFP, labeled with anti-GFP-Cy5 nanobodies, according to the distance to the coverslip. Download Copyright © 2020 Tejada-Arranz et al.2020Tejada-Arranz et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01173-20.9VIDEO S5Video S5, MOV file, 1.9 MB.3D visualization of superresolution imaging data corresponding to an example of representative bacteria in stationary growth phase. The yellow-red gradient is the membrane, labeled with WGA-AF555, according to the distance to the coverslip, and the blue-green gradient is RNase J-GFP, labeled with anti-GFP-Cy5 nanobodies, according to the distance to the coverslip. Download Copyright © 2020 Tejada-Arranz et al.2020Tejada-Arranz et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01173-20.10VIDEO S6Video S6, MOV file, 2.4 MB.3D visualization of superresolution imaging data corresponding to another example of representative bacteria in stationary growth phase. The yellow-red gradient is the membrane, labeled with WGA-AF555, according to the distance to the coverslip, and the blue-green gradient is RNase J-GFP, labeled with anti-GFP-Cy5 nanobodies, according to the distance to the coverslip. Download Copyright © 2020 Tejada-Arranz et al.2020Tejada-Arranz et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


Sulbactam (SUL)-durlobactam (SUL-DUR) is under development for the treatment of ABC infections.Durlobactam  is a novel β-lactamase inhibitor with broad-spectrum activity against Ambler class A, C, and D β-lactamases. Durlobactam restores the  in vitro activity of sulbactam (SUL) against members of the Acinetobacter baumannii-A. calcoaceticus complex (ABC). Sulbactam (SUL)-durlobactam (SUL-DUR) is under development for the treatment of ABC infections. Eighty patients with complicated urinary tract infection (cUTI), including acute pyelonephritis (AP), were randomized 2:1 to receive SUL-DUR at 1 g/1 g intravenously (i.v.) or placebo every 6 h (q6h) for 7 days and background therapy with imipenem-cilastatin (IMI) at 500 mg i.v. q6h to evaluate the tolerability of SUL-DUR in hospitalized patients. Patients with bacteremia could receive up to 14 days of therapy. SUL-DUR tolerability and the values of various pharmacokinetic (PK) parameters were determined. Efficacy was recorded at the test-of-cure (TOC) visit. SUL-DUR was well tolerated, with no serious adverse events (AEs) being reported. Headache (5.7%), nausea (3.8%), diarrhea (3.8%), and vascular pain (3.8%) were the most common drug-related AEs with SUL-DUR and were mostly of mild or moderate severity. The PK profile of DUR and SUL in hospitalized patients was consistent with observations in healthy volunteers. Overall success in the microbiological modified intent-to-treat (m-MITT) population was similar between the groups, as would be expected with IMI background therapy in all patients . SUL-DUR in combination with IMI was well tolerated in patients with cUTIs. The pharmacokinetics of SUL-DUR observed in hospitalized patients was similar to that observed in healthy volunteers. Durlobactam  is a novel β-lactamase inhibitor with broad-spectrum activity against Ambler class A, C, and D β-lactamases. Durlobactam restores the  Acinetobacter baumannii is identified by the Centers for Disease Control and Prevention and the World Health Organization to be a critical priority in need of new treatment options  and that has been associated with serious infections, including hospital-acquired bacterial pneumonia/ventilator-associated bacterial pneumonia (HABP/VABP), complicated urinary tract infections (cUTIs), bloodstream infections, and wound infections , a class A BLI, also exhibits antibacterial activity against ABC isolates; however, its use as monotherapy has been limited by increasing resistance   is a novel, diazabicyclooctenone β-lactamase inhibitor (BLI) that exhibits potent inhibition of class A, C, and D β-lactamases –18. In v–sistance . In precSUL-DUR) , 19, 20,SUL-DUR) , 22. SULAcinetobacter infections, the majority of whom are expected to be critically ill. Prior to dosing critically ill patients in a phase 3 study, assessment of the PK and tolerability of SUL-DUR in a phase 2 study with a hospitalized patient population was planned as a bridging strategy. This study evaluated the tolerability and PK of SUL-DUR in patients with cUTIs, including acute pyelonephritis (AP).Phase 1 clinical studies in healthy subjects evaluated the pharmacokinetic (PK) profiles of DUR after the administration of single and multiple ascending intravenous (i.v.) doses alone and in combination with SUL, the plasma and intrapulmonary concentrations of both components, and the drug-drug interaction potential between DUR and SUL –25. SUL-–n = 53) or placebo (n = 27). Two patients in the SUL-DUR group discontinued the treatment due to an adverse event (AE). One patient had moderate urticaria on day 3 of administration of the study drug that was self-limiting. The second patient was an 82-year-old patient whose serum creatinine concentration increased from 69 μmol/liter  on day 1 to 138 μmol/liter on day 3 and 109 μmol/liter on day 5. The study drug was discontinued on day 7, as the protocol did not allow for adjustment of the dose of imipenem-cilastatin (IMI) or SUL-DUR for changes in creatinine clearance, which would have been necessary to safeguard this elderly patient.Eighty patients were randomized to receive SUL-DUR  and a creatinine clearance of 94 ml/min . There was a gradual increase in the creatinine concentration, and on day 7 the creatinine concentration was 107 μmol/liter and the creatinine clearance was 61 ml/min. The study medication was discontinued due to reduced creatinine clearance on day 7, as the protocol did not allow for imipenem dose reductions.9/liter versus −1.42 × 109/liter for leukocytes and −4.96 × 109/liter versus −8.22 × 109/liter for neutrophils). However, the baseline leukocyte counts in these patients with infections were high, with the median in the SUL-DUR and placebo groups being 8.1 × 109/liter versus 6.75 × 109/liter. Moreover, no patients developed leukopenia or neutropenia. Mild changes in hepatic safety laboratory parameters from the baseline were observed at similar rates in patients in both groups. None of these changes were clinically significant or led to the discontinuation of therapy. No clinically meaningful changes in vital signs, electrocardiogram (ECG) findings, or physical findings were observed.No clinically meaningful changes in safety laboratory data were noted. A larger change from the baseline in the mean leukocyte and neutrophil counts was observed in the SUL-DUR group than in the placebo group at the late follow-up (LFU) visit (−0.24 × 10ss) and volume of distribution (Vss) of durlobactam were 10.3 liters/h and 31.6 liters, respectively. These values were similar to the mean clearance and volume of distribution estimates for sulbactam . The variability of the PK parameter estimates for clearance and volume of distribution were higher for sulbactam  than for durlobactam .The plasma concentrations of durlobactam and sulbactam in phase 2 study subjects were comparable over the 6-h sampling interval . The valThe overall rates of success in the microbiological modified intent-to-treat (m-MITT) population were similar in both groups, 36 (76.6%) patients in the SUL-DUR group and 17 (81.0%) patients in the placebo group, as would be expected with a background therapy with IMI in all patients. Overall success in the microbiologically evaluable (ME) population occurred in 36 (80.0%) patients in the SUL-DUR group and 17 (81.0%) patients in the placebo group.Proteus mirabilis isolate and two infected with IMI-NS Pseudomonas aeruginosa isolates) and in three of four (75%) patients in the placebo group (two infected with IMI-NS P. aeruginosa isolates and one of two [50%] infected with IMI-NS Klebsiella pneumoniae isolates).Seven patients presented with a baseline IMI-nonsusceptible  Gram-negative pathogen. For these patients, the overall success at the test-of-cure (TOC) visit occurred in three of three (100%) patients in the SUL-DUR group  administered every 6 h (q6h) via a 3-h i.v. infusion . This doPatients were enrolled at 20 clinical sites in Belarus, Bulgaria, Russia, and Ukraine between January 2018 and May 2018. The study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practices. The study protocol and amendments were approved by an institutional review board for each clinical site, and all patients provided written informed consent prior to any study procedure. This study was registered at ClinicalTrials.gov under identifier NCT03445195.This was a double-blind, randomized, placebo-controlled study to evaluate the tolerability and PK of i.v. SUL-DUR administered with IMI in patients with cUTIs, including AP, who were otherwise relatively healthy.Patients were randomized 2:1 to receive either SUL-DUR at 1 g/1 g i.v. or matching placebo. The treatments were reconstituted and diluted in 100 ml of 0.9% saline and then infused over 3 h every 6 h (q6h) for 7 days (28 doses). All patients received background therapy with IMI at 500 mg i.v. infused over 30 min q6h. Patients with bacteremia could receive up to 14 days of therapy. Randomization was stratified by baseline diagnosis (symptomatic cUTI versus AP), and at least 30% of the patients were required to have a diagnosis of AP at study entry.Male or female patients ages 18 to 90 years who were expected to require hospitalization and treatment with i.v. antibiotics for cUTI were eligible. A documented or suspected cUTI was defined based on the presence of at least two signs and symptoms, a urine specimen with evidence of pyuria, and the presence of at least one risk factor. Documented or suspected AP was defined by at least two signs or symptoms and a urine specimen with evidence of pyuria. Women of childbearing potential were required to have a negative pregnancy test before randomization and to use 2 highly effective methods of contraception until at least 30 days after the last dose of study drug. Men were required to use adequate contraception for at least 90 days after the last dose of study drug. Patients were excluded for the presence of any disease or condition that could confound the assessment of efficacy, including the use of any systemic antibiotic active against Gram-negative uropathogens for more than 24 h in the 72-h period prior to randomization.Tolerability was assessed from the incidence of treatment-emergent adverse events (TEAEs) and evaluation of changes from the baseline in the findings of clinical laboratory tests , 12-lead electrocardiogram (ECG), vital signs , and physical examination, including weight.Sparse sampling for PK analysis was completed, with samples being obtained predose on day 1, postdose of study drug on day 4 (±1 day) at the end of the infusion, and 0.5, 2, and 3 h after the end of the infusion (prior to the start of the next infusion). PK concentrations were analyzed using a validated liquid chromatography-tandem mass spectrometry assay .Clinical signs and symptoms were assessed at screening, on days 2 through 6, at the end of treatment , at the TOC visit (7 days post-EOT), and at the late follow-up (LFU) visit (7 days post-TOC visit).No formal sample size calculation was performed. At least 80 patients were expected to be randomized 2:1 to SUL-DUR or placebo. The intent-to-treat (ITT) population included all randomized patients. The modified intent-to-treat (MITT) population included patients who met the criteria for the ITT population and received any study drug and was used as the population for the primary analysis of tolerability.kel) could not be estimated, half-life, steady-state volume of distribution (Vss), and accumulation index were not reported. The PK parameters evaluated included half-life, the time after dosing at which the maximum concentration was observed (Tmax), the maximum observed concentration measured after dosing (Cmax), the minimum observed concentration measured after dosing (Cmin), kel, Vss, the steady-state clearance (CLss), the steady-state area under the concentration-versus-time curve from the dosing time to the dosing time plus τ (AUC0–τ) , and the accumulation index. The PK parameters were reported using descriptive statistics.The values of the pharmacokinetic parameters in the PK population (the MITT population with at least one plasma PK sample drawn) were estimated by noncompartmental analysis, which was completed using Phoenix PK software (WinNonlin), version 8.1. The area under the plasma DUR or SUL concentration-versus-time curves (AUC) was calculated using the linear up-log down method. Where the first-order elimination rate constant (


Serum creatinine, total cholesterol, fasting blood glucose and HbA1c were also measured. One-way ANOVA, Kruskal-Wallis Test, Mann-Whitney U Test corrected by Bonferroni, Pearson Correlation Analysis and Spearman Correlation Coefficient Analysis were used for data analysis.Plasma ephrin-A1 and VEGF165 in human plasma was 34.00 ± 42.55 pg/ml, with no statistical difference among the three groups. There was no correlation between ephrin-A1 and VEGF165 in human plasma, but there was a correlation between plasma ephrin-A1 and duration of diabetes.Ephrin-A1 expression could be detected in human plasma with an average of 1.52 ± 0.43 (mean ± SEM) ng/ml. In DR subjects, the plasma ephrin-A1concentration was 3.63 ± 4.63 ng/ml, which was significantly higher than that of the other two groups . The expression of VEGF165 expression in patients with diabetic retinopathy compared to the other two groups, suggesting that changes of plasma ephrin-A1 may be a more sensitive biomarker than plasma VEGF165 in detecting diabetic retinopathy.Plasma ephrin-A1 was highly expressed in patients with diabetic retinopathy, and there was no difference of plasma VEGF It is estimated that there are 382 million diabetic patients worldwide, and about one third of these patients have diabetic retinopathy (DR), part of them have diabetic macular edema. DR causes visual impairment in about 37 million people around the world . About 6165 appears to be the most abundant and potent isoform in inducing neovascularization, followed by VEGF121 and VEGF189 [In the pathogenesis process of DR, endothelial-derived growth factor (VEGF) is a major cytokine that promotes neovascularization, which leads the destruction of blood retinal barrier and indirectly promoting the progress of DR. Several spliced isoforms of VEGF present in human body with 121, 145, 165, 183,189, and 206 amino acids in length [ VEGF189 . In addi VEGF189 . However VEGF189 . In addiEphrins and the Eph receptors have been identified as key regulators of angiogenesis . Class AOver 40 years of age.Clinical diagnosis of primary angle-closure suspect, diabetes mellitus and DR.Patients agreed to provide informed consent.This study was approved by institutional ethics committee in the Fenghua Hospital of Traditional Chinese Medicine. Informed consents in written format to publish these date were collected from each patient, who agreed and signed the consent to participate statement. Primary angle-closure suspect was inclusion criteria, because there were no reports about the association between ephrin-A1 and primary angle-closure suspect, or VEGF and primary angle-closure suspect in the literature. In addition, there is no vascular changes or cytokine changes that are associated with angiogenesis in primary angle-closure suspect. The recruited patients were enrolled between August 1, 2018 and October 31, 2019 with the following inclusion criteria:The definition of primary angle-closure suspect was in accordance with the International Society of Geographical and Epidemiologic Ophthalmology criteria . DiabeteThe subjects with any diseases that presented neovascularization such as kidney disease and cancer, or any of those subjects received anti-VEGF therapy or operations were excluded.A total of fifty-five patients from Fenghua District were recruited. Nineteen patients with primary angle-closure but without diabetes were enrolled in the non-DM group. Sixteen patients with primary angle-closure and diabetes were enrolled in DM group. Twenty patients with DR were enrolled in DR group, about 35% of patients with proliferative diabetic retinopathy.After routine physical and eye examinations, electrocardiogram, clinical laboratory tests including liver and kidney function tests, routine ophthalmic examinations, fundus photograph and fluorescein angiography were performed.The averaged duration time of type 2 DM in the diabetic group was 46 ± 18.5 months. The averaged duration time of type 2 DM with DR was 118 ± 49 months.The blood samples were collected using ethylenediaminetetraacetic acid (EDTA) as an anticoagulant from the peripheral vein of each patient and transferred to Department of Clinical Laboratory, which is a section of Fenghua Hospital of Traditional Chinese Medicine. Samples were centrifuged immediately for 15 min at 1000 g. Then, plasma was collected and stored at − 80 °C before enzyme-linked immunosorbent assay (ELISA) measurement.All individuals were informed of the purpose of the study and their informed consent was obtained. This study followed the tenets of the Declaration of Helsinki and was approved by the ethics committee of the institutional Fenghua Hospital of Traditional Chinese Medicine.165 was measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit , following the instructions. Results were obtained by a multifunction microplate reader . In addition, 6 ml venous blood of subjects was taken for measurement of fasting blood glucose, HbA1c, serum creatinine, and total cholesterol levels.Ephrin-A1 was measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit  and VEGF165, fasting blood glucose levels, HbA1c, serum creatinine and total cholesterol were examined by Spearman’s Correlation Analysis. Correlation between ephrin-A1 and duration of diabetes was performed by Pearson Correlation Analysis. P < 0.05 was considered statistically significant.Statistical analyses were performed using SPSS 18.0 . The data were shown as mean ± SD. After inspecting the distribution of the data, we assessed statistical significances with One-way ANOVA, Kruskal-Wallis Test, Mann-Whitney U Test corrected by Bonferroni. Correlations between any two of parameters including age, BMI, plasma ephrin-A1, VEGF165, serum creatinine and total cholesterol among the three groups of subjects. No relationship between Ephrin-A1 and VEGF165 in human plasma had been found. There were differences in fasting blood glucose and HbA1c among the three groups. Compared to the non-DM subjects, DM patients and DR patients had higher fasting blood glucose and HbA1c (P < 0.05).Clinical and laboratory data were shown in Table P < 0.05) ng/ml. The concentration of ephrin-A1 in the DR subjects was significantly higher than that of the other two groups  and with Serum creatinine , but was not correlated with age, BMI, serum VEGF165, total cholesterol, fasting blood glucose and HbA1c in the DR patients, the DM subjects, and the non-DM subjects or all subjects.Plasma ephrin-A1 was correlated with the duration of diabetes (Interaction between EphA receptor tyrosine kinases (RTKs) and ephrinA ligands is necessary for inducing of maximal neovascularization by VEGF . EphA2 RIn this study, ephrin-A1 concentrations in the plasma of non-diabetic, diabetic and DR patients were measured in a cohort of subjects. The averaged ephrin-A1 concentration in human plasma was 1.52 ± 0.43 (mean ± SEM) ng/ml, indicating ephrin-A1 was expressed in human plasma. We found ephrin-A1 concentration in the DR group was higher than that in the non-diabetic and the diabetic groups, suggesting ephrin-A1 may be involved in the development of DR. There is evidence showed that ephrin-A1 inhibits VEGF-induced intracellular signaling and suppresses retinal neovascularization and blood-retinal barrier breakdown . Previou165 in these patients. We did not find difference in plasma VEGF165 among the three groups. This result was inconsistent with a previous report that the plasma VEGF165 concentration elevated in DR patients compared to non-DR controls [165 in the systemic circulation in DR patients.We also measured the plasma concentrations of VEGFcontrols . FurtherThis study is the first report that ephrin-A1 is presented in circulation. Furthermore, ephrin-A1 expression is elevated in DR patients, suggesting it may be involved in the pathogenesis of DR. Whether it may become a biomarker for the disease deserves further studies. Where does the increase ephrin-A1 come from and the mechanism of ephrin-A1 upregulation requires further research.


The pressure-dependent capacitance variation in our device was explained based on the change in the contact-area formed between the multi-filament fiber electrodes and the PVDF-HFP/IL film. To demonstrate the applicability and scalability of the sensor device, a 3 × 3 pressure sensor array was fabricated. Due to its matrix-type array structure and capacitive sensing mechanism, multi-point detection was possible, and the different positions and the weights of the objects could be identified.Textile-based pressure sensors have garnered considerable interest in electronic textiles due to their diverse applications, including human–machine interface and healthcare monitoring systems. We studied a textile-based capacitive pressure sensor array using a poly(vinylidene fluoride)-co-hexafluoropropylene (PVDF-HFP)/ionic liquid (IL) composite film. By constructing a capacitor structure with Ag-plated conductive fiber electrodes that are embedded in fabrics, a capacitive pressure sensor showing high sensitivity, good operation stability, and a wide sensing range could be created. By optimizing the PVDF-HFP:IL ratio (6.5:3.5), the fabricated textile pressure sensors showed sensitivity of 9.51 kPa Electronic textiles (e-textiles) have recently garnered considerable interest in the field of wearable electronics due to the diverse applications that are enabled by integrating them into various sensor devices ,2,3, tri−1 under 10 kPa [TFSI]) IL  were mixed at different weight ratios and stirred for −1 h. The weight ratios of PVDF-HFP:IL varied as 5:5, 6:4, and 6.5:3.5. To fabricate a freestanding PVDF-HFP/IL film, which is used as a dielectric layer in the textile pressure sensor, a 3 cm × 3 cm bare glass substrate was prepared and thoroughly cleaned with acetone, isopropanol alcohol, and deionized water, then dried with dry nitrogen. The PVDF-HFP/IL solution was dropped on the cleaned glass substrate using a micropipette with a dropping volume of −10 μL. Next, to cure the film, a thermal annealing at 40 °C was carried out for 12 h. After the curing, the film was detached from the glass substrate. The average diameter of the fabricated PVDF-HFP/IL film was −0.5 cm. To fabricate the pressure sensor, Ag-plated conductive nylon fibers  was stitched on both the top and bottom polyester fabric sheets, which had dimensions of 5 cm × 5 cm. A double-sided thermoplastic adhesive film  was then bonded to the bottom polyester fabric sheet by applying 2.56 kPa of pressure at 110 °C for 10 min. To expose the Ag-plated fiber electrode, which was located at the center region of the fabric sheet, the center region of the adhesive film was cut to a dimension of −0.5 cm × 0.5 cm. Then, the free-standing PVDF-HFP/IL film was placed on top of the Ag-plated fiber electrode. Next, a top fabric sheet having Ag-plated fiber electrode was covered, and finally the sample was hot pressed for 1 min by applying 2.56 kPa of pressure at 60 °C to bond the fabric sheets together.The fabrication process of PVDF-HFP/IL film is shown in The surface morphology of the PVDF-HFP/IL film was analyzed using a field-emission scanning electron microscope (FESEM) . To measure the pressure-sensing characteristics, a force gauge  was utilized. The measured pressure range was 0–100 kPa. The characterizations of the pressure sensor were carried out using a probe station  connected to an LCR meter  and a semiconductor parameter analyzer . The capacitance value of the pressure sensor was measured with an applied voltage of 1 V at frequencies of 0.1–100 kHz.The optical and FESEM images of the PVDF-HFP/IL film are shown in 0) value of the film, PVDF-HFP/IL films with different ratios were prepared. 0 as a function of PVDF-HFP:IL ratio. When the PVDF-HFP:IL ratio was varied as 6.5:3.5, 6:4 and 5:5, the C0 increased to 0.062 nF, 0.85 nF and 9.32 nF, respectively. The increased C0 can be attributed to the larger amount of ILs in the PVDF-HFP/IL film, which contributed to the EDL formation at the interfaces with the top and bottom fiber electrodes. According to the Stern theory [0 value decreased from −25 nF to 0.062 nF when the frequency was increased from 100 Hz to 100 kHz. In our study, because the initial capacitance value directly affected the pressure sensitivity (S) as shown in the following equation:0 value. As shown in 0 value was only changed by 2.7% after the bending test, indicating that the PVDF-HFP/IL film had good mechanical stability.To investigate the effects of PVDF-HFP:IL ratio on the initial capacitance  was largely dependent on the PVDF-HFP:IL ratio, and particularly, the sensors fabricated with PVDF-HFP:IL ratio of 6.5:3.5 exhibited the highest capacitance change, while those fabricated with PVDF-HFP:IL ratios of 5:5 and 6:4 showed much reduced capacitance variations (1) and 20–100 kPa (S2). When the PVDF-HFP:IL ratio was 6.5:3.5, the S1 and S2 were 9.51 kPa−1 and 0.69 kPa−1, respectively. However, as the IL weight ratio was increased, the S1 and S2 decreased substantially. One of the main reasons for the reduced sensitivity was the high C0 values of the film  by the applied pressure decreased, which can be also attributed to the higher C0 values at lower frequencies (2) was relatively low when the frequency was 0.1–10 kHz, limiting the detection range of the sensor. In overall, the results suggest that a PVDF-HFP:IL ratio of 6.5:3.5 and an operation frequency of 100 kHz are the most suitable to achieve high-pressure sensitivity and a wider pressure-detection range.Additionally, because the capacitance of PVDF-HFP/IL film is also varied by the frequency, the frequency-dependent pressure-sensing characteristics were evaluated. As shown in quencies b. More iε is permittivity of the dielectric, A is the area, and d is the thickness of the dielectric film. Then, by applying an external pressure, the area and the thickness of the dielectric film can be changed, thereby increasing the capacitance. In our device structure, it was suggested that although the thickness of PVDF-HFP/IL film was decreased by pressure, the increase of contact area made between the multi-filament fiber electrodes and the PVDF-HFP/IL film was considered as the dominant mechanism for the capacitance variation [In the PVDF-HFP/IL textile pressure sensor, the two multi-filament Ag-plated fiber electrodes and the PVDF-HFP/IL dielectric film formed a capacitor. Therefore, the capacitance can be expressed by the following equation:ariation , particuThe response characteristics of the pressure sensor are also important parameters for determining the applications of the sensor. n , and the row fiber electrodes were designated as Yn  (n (n = 1–9). Because the 3 × 3 pressure sensor array operated based on the capacitance variation, a multi-point pressure detection was possible. To demonstrate the multi-point pressure detection, one to three separate objects were placed at different sensing points on the sensor array  a. As shoor array c–f. The In our study, we demonstrated a textile-based capacitive pressure sensor using a PVDF-HFP/IL composite film and multi-filament Ag-plated fiber electrodes. Using an optimized PVDF-HFP:IL ratio, we created a textile pressure sensor that exhibited a high pressure sensitivity, a wide detection range, and good operation stability. The PVDF-HFP:IL ratio had significant effects on the sensitivity and detection range of the sensor. The pressure-dependent capacitance variation was explained based on the change in the contact area, which was formed between the multi-filament fiber electrodes and the PVDF-HFP/IL film. To demonstrate the applicability and scalability of the PVDF-HFP/IL-based sensor, we used a 3 × 3 pressure sensor array; due to its matrix-type array structure and the capacitive-type sensing mechanism, identification of the different positions and weights of the objects was possible.


In a first proof-of-concept study, we could show that IA was effective to reduce ß2AR-AB and led to improvement of various symptoms. (2) Five of the ME/CFS patients who had clinical improvement following treatment with a five-day IA were retreated in the current study about two years later with a modified IA protocol. The severity of symptoms was assessed by disease specific scores during a follow-up period of 12 months. The antibodies were determined by ELISA. (3) The modified IA treatment protocol resulted in a remarkable similar clinical response. The treatment was well tolerated and 80–90% decline of total IgG and ß2AR-AB was achieved. Four patients showed a rapid improvement in several clinical symptoms during IA therapy, lasting for six to 12 months. One patient had no improvement. (4) We could provide further evidence that IA has clinical efficacy in patients with ME/CFS. Data from our pilot trial warrant further controlled studies in ME/CFS.(1) Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex neuroimmunological disease. There is evidence for an autoimmune mechanism for ME/CFS with an infection-triggered onset and dysfunction of ß Furthermore, it is accompanied by a variety of other symptoms that are related to immunological and autonomous dysfunction . With anpatients ,12,13. Tpatients ,15. A reith pain . Resultsith pain ,18. Apprituximab .2AR-AB were elevated [2AR-AB decreased rapidly during IA treatment and it was still significantly lower than the pretreatment level after six months. The frequency of memory B cells significantly decreased, whereas the frequency of plasma cells increased after a five-day IA cycle. A rapid improvement of symptoms was reported by 70% of patients already during IA. Three of these patients had long lasting improvement for more than 12 months and four patients had short improvement. However, two patients had a marked worsening of symptoms during IA and could not receive the fifth IA.An effective treatment option for autoantibody-mediated diseases is immunoadsorption (IA). By using specific adsorbers, the plasma concentration of immunoglobulin G (IgG) can be quickly and efficiently reduced . Clinicaelevated . Five cyHere, we present data of a conformational trial. Patients who had responded to the first IA (IA1) were retreated with a modified IA protocol (IA2) about two years later. Here, IA cycles were given with longer intervals.2AR-AB levels, and an infection-triggered disease onset.For the conformational trial, we adjusted the treatment protocol IA2, see . Five of® columns , a broadband-immunoadsorber containing synthetic peptide-GAM® as ligand capable of binding IgG and immune complexes independent from their antigen specificity and, thus, useful for the removal of autoantibodies. Patients received 10 g polyclonal immunoglobulin substitution intravenously  to restore IgG plasma levels after the final IA session. The study was approved by the Ethics Committee of Charité - Universitätsmedizin Berlin  in accordance with the 1964 Declaration of Helsinki and its later amendments. All of the patients gave written informed consent.During the IA1 study, we learned that repeated IA can worsen fatigue. Four patients had worsening of fatigue towards the end of treatment despite improvement of other symptoms. To improve the tolerability of the treatment, we extended the treatment period and reduced the dose of IgG replacement. IA was performed at day one, two, four, six, and eight  using Gl1AR-/ß2AR-AB and M3AR-/M4AR-AB were determined using ELISA technology by CellTrend GmbH , as in our previous study [ßus study . Total sWe assessed the presence and severity of symptoms, as described in our previous study . In shorp-value of <0.05 was considered to be statistically significant.We conducted statistical data analyses using GraphPad Prism version 6.0 software similarly to the first proof-of-concept study . In shor1/ß2-AR-AB and M3/M4-AR-AB (except patient 8 only ß2). Bell disability scale indicating the severity of disease ranged from 30–75 (median of 45) and the median was significantly higher than before the first IA study in 2016 . As expected, the absolute IgG levels were strongly reduced already after the first IA with a minimum IgG level after the fourth IA . The absolute level of autoantibodies decreased in a comparable way, as shown in n = 4), and immune-associated symptoms of sore throat, flu-like symptoms, and painful lymph nodes  and from concentration impairment grade 4–10, muscle pain  in ME/CFS ,31, the Our study was designed to get further evidence for efficacy and tolerability prior to performing a consecutive RCT. We have evidence for similar clinical and immunological efficacy of this schedule.In summary, the current study provides further evidence that IA is effective in ME/CFS. This result warrants further studies of repeat IA therapy to maintain clinical remission, as shown for other autoimmune diseases . Another


In this experiment, P. duplex AARL G060 generated low (9.03 mg/L) and high (42.41 mg/L) concentrations of BioMnOx. The precipitated BioMnOx was observed by scanning electron microscopy (SEM), and the elemental composition was identified as Mn and O by energy-dispersive x-ray spectroscopy (EDS). The potential for BioMnOx to act as a catalyst in the degradation of paraquat was evaluated under three treatments: (1) a negative control (deionized water), (2) living alga with low BioMnOx plus hydrogen peroxide, and (3) living alga with high BioMnOx plus hydrogen peroxide. The results indicate that BioMnOx served as a catalyst in the Fenton-like reaction that could degrade more than 50% of the paraquat within 72 h. A kinetic study indicated that paraquat degradation by Fenton-like reactions using BioMnOx as a catalyst can be described by pseudo-first and pseudo-second order models. The pH level of the BioMnOx catalyst was neutral at the end of the experiment. In conclusion, BioMnOx is a viable and environmentally friendly catalyst to accelerate degradation of paraquat and other toxic chemicals.Paraquat is a non-selective fast-acting herbicide used to control weeds in agricultural crops. Many years of extensive use has caused environmental pollution and food toxicity. This agrochemical degrades slowly in nature, adsorbs onto clay lattices, and may require environmental remediation. Studies have shown that biosynthesized manganese oxide (BioMnO Pediastrum duplex AARL G060.BiOMnOx catalyst generated from living  Non-selective herbicides, such as glyphosate and paraquat, were developed based upon their antagonistic effects on non-specific species of weeds and crops. Paraquat  transforms electron flow from the photosystem and inhibits reduction of oxidized nicotinamide adenine dinucleotide phosphate (NADPC) during photosynthesis . IntensiPrevious studies reported paraquat contamination in many countries worldwide . A paraqTo protect human health from the toxic effects of paraquat, removal is required. Biological methods may provide limited paraquat degradation  is a conventional method for paraquat degradation based on the generation of highly reactive and non-selective hydroxyl radicals (∙OH) . The higxidation . Since t2 and H2O (eq. 3). However, Fenton’s reaction requires acidic conditions  with high oxidative potential. These radicals attack organic matter in wastewater (eq. 2) and generate oxidation intermediates which are further attacked by hydroxyl radicals to produce COparaquat , and the2O2 decomposition , as catalysts for Hposition . The reaDesmodesmus sp. WR1 and P. duplex AARL G060 increase dissolved oxygen content due to microalgal photosynthesis  to As(V), which is a preliminary step for As removal  degradation. The BioMnOx was prepared using living microalga P. duplex AARL G060 via a biological reaction process. The results of this study demonstrate the feasibility of applying BioMnOx for removal of harmful agents in the environment.Recent research demonstrated the application of BioMnOreatment . Another removal . The remcatalyst . In addiolecules . For exazed MnOx . HoweverP. duplex AARL G060 was obtained from the Applied Algal Research Laboratory (AARL), Department of Biology, Faculty of Science, Chiang Mai University, Thailand. P. duplex can generate BioMnOx at high concentrations of Mn, and so this algal strain was selected for use in this study. An axenic culture of microalgal stock was maintained in Jaworski’s Medium under the following condition: 72.51 μE⋅m–2⋅s–1 light intensity from a light emitting diode, 25°C ambient temperature, and continuous shaking at 130 rpm.Living green microalga Chromatographic separation of paraquat was performed by application of high-performance liquid chromatography (HPLC). Paraquat (98% pure) was purchased from MilliporeSigma. Acetonitrile of HPLC grade was purchased from Merck KGaA. All other reagents were analytical grade.Paraquat stock solution, at a concentration of 1,000 mg/L, was prepared by dissolving 0.6910 g of paraquat in 1,000 mL of DW. The paraquat stock solution was then diluted to the desired concentration using simple dilution methods .x using microalgae.Water samples obtained from the surface of a rehabilitated lignite coal-mine reservoir in the northern part of Thailand . Water samples were collected in November and December 2017. Some physico-chemical parameters of the water sample were published elsewhere . High-deP. duplex AARL G060 were cultivated in water samples obtained from the rehabilitated reservoir. The microalga was cultivated with nutrients containing NaNO3 (0.09438 g/L), KH2PO4 (0.02606 g/L), CaHCO3 (0.0159 g/L), and MgSO4.7H2O  to compare to the control (modified medium in DW without paraquat). All treatments were performed in triplicate at ambient temperature and 111.81 mE m–2 s–1 light intensity using LED illumination. The microalga density was determined daily by measuring the OD665 using a Thermo Fisher ScientificTM GENESYSTM 20 Visible Spectrophotometer, and the pH value of the cultures was measured using a Starter 3100 pH Bench . Living microalga cultures in flasks were shaken three times per day by hand to confirm that all algal cells were suspended and to prevent the dissolution of oxygen due to agitation which interferes with Mn oxidation during photosynthesis.Living green microalga x nanoparticles were prepared via photosynthesis of P. duplex AARL G060 and designated as low or high concentration. At low BioMnOx concentration (LA-Low-BioMnOx), the microalga was inoculated in 300 mL of natural wastewater containing a Mn concentration of 20 mg/L. For high BioMnOx concentration (LA-High-BioMnOx), living microalga was inoculated in 900 mL of natural wastewater containing a Mn concentration of 60 mg/L, triple the concentration of low BioMnOx. After six days of cultivation, cell pellets containing BioMnOx were collected by centrifugation at 3,500 rpm for 20 min. Next, samples were washed twice with a phosphate buffer at a pH of 8.00. Then washed with DW to remove residual Mn ions from the microalgal cells. The amount of precipitated BioMnOx that remained on the cell pellets was dissolved with 10 mL of 1 mM EDTA at a pH of 3.40 and analyzed using the same method applied to Mn concentrations in the supernatant .Manganese concentrations in this experiment were assigned the highest Mn concentrations found in the rehabilitated reservoir 20 mg/L, . The Bioernatant . The Biox catalyst produced by P. duplex AARL G060 was collected by centrifugation at 4,000 rpm. Cell pellets were fixed with 2.5% glutaraldehyde in 0.1 M of phosphate buffer at a pH of 8.00 overnight at 4°C. The samples were then washed with a phosphate buffer at a pH of 8.00. After that, the samples were dehydrated with an ethanol concentration series. Next step, the sample was mounted on stubs and thereafter gold-sputtered  the control , 2) the culture containing the living P. duplex AARL G060 with low BioMnOx (LA-Low-BioMnOx), and 3) a culture containing living P. duplex AARL G060 with high BioMnOx (LA-High-BioMnOx). Paraquat degradation was performed in 150 mL of culture in a 250 mL Erlenmeyer flask. Then, 13.5 mL of H2O2 was added to LA-Low-BioMnOx and LA-High-BioMnOx treatments following the method of BioMnO0 is the initial paraquat concentration and Ct is the concentration at time t.where CThe HPLC was operated under the following conditions: the HPLC column was a VertiSep UPS-C18 analytical column  maintained at a temperature of 30°C. The mobile phase consisted of 0.14 mol sodium chloride and acetonitrile . All samples were eluted at a flow rate of 1 mL/min. The column eluent was monitored by HPLC at ambient temperature (25°C) using a Diode-Array Detection (DAD) detector . The wavelength was fixed at 257 nm. Calibration curves for the analysis were established with a standard solution of paraquat.TM Excel 2016.All of the graphs and statistical analyses were completed using MicrosoftP. duplex AARL G060 was cultivated in a medium containing of N, P, C, and Mg to measure its growth . The oxide form of the Mn (MnOx) was achieved when the pH level in culture was raised. The results indicate that P. duplex AARL G060 grow well in both LA-Low-BioMnOx and LA-High-BioMnOx treatments  treatment maintained fairly stable paraquat concentrations. The pH level was above 5 from the initial time to the last hour of contact time :A kinetic study of paraquat degradation in a Fenton-like reaction using BioMnOx as a catalyst was performed, and the pseudo-first order and pseudo-second-order kinetic models were applied to model the kinetics of paraquat degradation under different BioMnO0 and Ct represents are initial paraquat concentration and concentration at time t, k1 is the rate of degradation (constant) and the time t. Events for the pseudo-second-order model were described by Eq. (8):where C2 is the constant rate of degradation.in which, kx-H2O2 treatment was fast during the initial period of 0–6 h, and the highest R2 value for this treatment was described as pseudo-first order and pseudo-second order kinetic model . This treatment could be described as pseudo-first order model because the k1 value was 0.2012 h–1. Higher constant rates revealed in this treatment demonstrated paraquat degradation efficiency higher than that of the LA-Low-BioMnOx-H2O2 treatment. Meanwhile, in a later period (6–72 h), the reaction of both treatments showed low degradation rates, which indicates a slower reaction.The results of the kinetic study indicate that paraquat degradation in LA-Low-BioMnOic model . This fiP. duplex AARL G060. The growth of microalga in LA-Pq treatment decreased with time. A previous study reported that paraquat accepted electrons from photosystem I, thus preventing electron transport to NADPH, and this action may block photosynthesis  stress and generated up to 240 mg/L of Mn oxide in 6 days. The BioMnOx coupled with peroxymonosulfate (PMS) activation degraded 99.5% of 2,4-dimethylaniline within 80 min :The application of chemical paraquat degradation methods discovered that the chemical process is a Fenton-like reaction that relies on metals, such as cerium, chromium, manganese, cobalt, and copper, to directly decompose Hinto ∙OH . A bioloThen, a Fenton-like reaction, involving Mn ions proceeded as shown in Eqs. (10–13):2+ and Mn4+ through intermediate Mn3+ species should allow the Mn-catalyzed Fenton-like activation of H2O2. The advantages of MnOx oxidation have received intensive attention due to the large application area and environmental friendliness (Interconversion between Mnndliness .x treatment easily degraded paraquat in first period (0–6 h). The kinetics of the Fenton-like reaction during this initial period was described as pseudo-first and pseudo-second order kinetic models. During this period, the Fenton-like reaction used Mn ions which could increase reaction rates. During the last period (6–72 h), this treatment showed slower reaction rates and a decrease in paraquat degradation. The reason for this phenomenon is unclear but may be explained as follows. First, when P. duplex AARL G060 synthesized BioMnOx, Mn particles were generated both intracellularly and extracellularly  of this treatment showed a high kinetic rate constant (0.2012 h–1). This degradation percentage of paraquat in LA-High-BioMnOx was higher than that of LA-Low-BioMnOx treatment during this period. During the last period (6–72 h), the kinetic rate constant was lower than the first period and also indicated that degradation rates stabilized in this phase. Even for the amount of BioMnOx catalyst loading observed, the results are consistent with previous studies that attributed catalyst loading to an increase in the active substances; which, in turn, form more active radicals to contact the target pollutant (3O4-MnO2 nano-complex removed up to 96.8% of acid orange 7. The MnO catalyst performed better and degraded more toxin than either Fe3O4 or MnO2 alone.The results of the LA-High-BioMnOollutant , consistollutant . Moreoveollutant . In addix on microalgal cell surfaces are shown in x nanoparticles varied according to different Mn ion concentrations, microalgae concentrations, and cultivation time. Other than using viable microalgal cells, cell free extracts were also possible to biosynthesize for nano MnO (Paraquat degradation efficiency depended on the dispersions of the catalyst. The highly aggregated BiOMnOnano MnO . In Kumax-H2O2 treatment after 72 h decreased. Regeneration of the catalyst could help degradation efficiency remain nearly constant. The observed paraquat degradation by the Fenton-like LA-High-BiOMnOThe HPLC profiles showed rapid disappearance of the peak corresponding to paraquat, accompanied by the appearance of new peaks. Although degradation products of paraquat were not evaluated in this study, degradation products from AOPs were suggested elsewhere. The degradation products may correspond to demethylation products such as 4,40-bipyridine and monopyridone and hydroxylation or oxidative ring cleavage products such as 4-carboxy-1-methyl-pyridinium ion, 4-picolinic acid, and hydroxyl-4-picolinic acid, in accordance with paraquat degradation by UV-ozonation , or degrx as a catalyst to remove contaminants from real-life full-scale wastewater provides a viable environmentally friendly alternative technology for the treatment of industrial wastewater.The Fenton-like reactions using BiOMnOP. duplex AARL G060 was found to be a potent strain of living green microalgae for BioMnOx production. The BioMnOx served as a biocatalyst that degraded 54.64% of the paraquat in aqueous solution, and the pH level of this operation was above 5. This result demonstrates that BioMnOx is an environmentally friendly alternative catalyst to remove toxins in wastewater. The insights gained from this experiment will be used to develop better treatments for degradation of toxins and remediation of wastewater.Based on the results of this study, All datasets generated for this study are included in the article/All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Asthma patients experience impairments in health-related quality of life (HRQL). Interventions are available to improve HRQL. EQ-5D-5L is a common generic tool used to evaluate health interventions. However, there is debate over whether the use of this measure is adequate in asthma patients.We used data from 371 asthma patients participating in a pulmonary rehabilitation (PR) program from the EPRA randomized controlled trial. We used four time points: T0 randomization, T1 start PR, T2 end PR, T3 3 months follow-up. We calculated floor and ceiling effects, intra-class correlation (ICC), Cohen’s d, and regression analysis to measure the sensitivity to changes of EQ-5D-5 L ) and the disease-specific Asthma Quality of Life Questionnaire (AQLQ). Furthermore, we estimated the minimally important difference (MID). Based on the Asthma Control Test (ACT) scores, we defined three groups: 1. ACT-A (ACT> 19) controlled asthma, 2. ACT-B (14 < ACT≤19) not well-controlled asthma, and 3. ACT-C (ACT≤14) very poorly controlled asthma.Only the EQ-5D index showed ceiling effects at T2 and T3 (32%). ICC (between T0 and T1) was moderate or good for all measures. Cohen’s d at T2 and T3 was better at differentiating between ACT-A and ACT-B than between ACT-B and ACT-C. The EQ-5D index showed moderate effect sizes (0.63–0.75), while AQLQ showed large effect sizes . VAS was responsive to pronounced positive and negative ACT changes in every period, and AQLQ mostly to the positive changes, whereas the EQ-5D index was less responsive. We estimated a MID of 0.08 for the EQ-5D index, 12.3 for VAS, and 0.65 for AQLQ.All presented HRQL tools had good discriminatory power and good reliability. However, EQ-5D-5 L did not react very sensitively to small changes in asthma control. Therefore, we would suggest using supplementary measures in addition to EQ-5D-5 L to evaluate asthma-specific interventions more comprehensively.https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00007740. The registration took place prospectively.German Clinical Trial Register, DRKS00007740 (date of registration: 05/15/2015),  Asthma is a respiratory disease characterized by chronic inflammation of the airways. Asthma patients experience cough, wheeze, and shortness of breath in varying intensity and frequency . This syTwo groups of HRQL assessment tools exist, disease-specific and generic ones. Disease-specific assessment tools are developed for specific diseases. They mainly focus on the impact of disease symptoms and the related consequences, but might also cover aspects of disease-associated impairments in social participation or emotional and general wellbeing. They enable comparisons between patients at different stages of the same disease and help to monitor disease development. In contrast, generic assessment tools can be applied across different diseases because they focus on impairments in general health-related aspects of life. Thus, comparisons between different disease areas or with the general population become possible. However, they might not always fully capture HRQL impairments in the context of disease-specific symptoms, especially in the early stages of a disease .One of the most commonly used generic assessment tools is the EQ-5D-5L from the EuroQol group , which iThere is debate over whether the use of the generic EQ-5D is adequate in asthma patients. Whalley et al. The three-level version has already raised some concerns, e.g., its inefficient ability to differentiate between different levels of asthma control  or that Therefore, our aim is to investigate whether the EQ-5D-5L is suited to measure HRQL in asthma patients in a longitudinal setting, whether it is reliable, and if it is responsive to changes in asthma control, compared with the established disease-specific Asthma Quality of Life Questionnaire (AQLQ). Furthermore, we aim to provide a MID value for the five-level version for asthma patients, which has not to our knowledge been provided in previous studies.We used data from the EPRA study, a randomized controlled trial (RCT) using a wait-list control group assessing the effectiveness of PR among asthma patients . After approval for rehabilitation (T0), patients were randomized to the intervention group (IG) or control group (CG). The IG started the 3-week PR 4 weeks after randomization , whereas the CG started PR 5 months after randomization (T3). Further details of the study have been published elsewhere . We asseWe assessed disease severity and HRQL using the following measures:The ACT is a self-administered questionnaire to evaluate asthma control . It contThe standardized version of the AQLQ is an asthma-specific HRQL assessment tool containing 32 questions in four domains  , 23. TheThe EQ-5D-5L is a generic HRQL measure from the EuroQol group , which eThe GROC is a rating scale with 15 categories assessing the self-reported change in global health. Patients with improvement and deterioration are symmetrically distributed around zero , 27, witAll analyses were performed with SAS , and p-values of 0.05 or less were considered statistically significant. We looked at floor and ceiling effects at every time point, defined as > 15% of the patients reaching the best/worst HRQL score . FurtherKnown-group validity (Cohen’s d) is used to evaluate the ability of the HRQL tools to differentiate between disease severity groups. Cohen’s d was assessed as the mean adjusted differences in HRQL scales between the ACT groups, divided by their pooled standard deviation at T2 or T3. We adjusted for group (IG/CG), age, sex, smoking status, body mass index (BMI), and employment status before PR (yes/no) to compensate for changes not originating from a change in ACT. Cohen’s d was considered small between 0.2 and 0.5, moderate from 0.5 to 0.8, and large above 0.8 .To estimate the reliability of the HRQL questionnaires, we evaluated ICC   between To estimate the responsiveness of HRQL scales associated with a change in ACT, we conducted different regression analyses for each HRQL scale. The dependent variable was the HRQL change score (ΔHRQL) in three periods . The independent variables were ACT change (ΔACT) in five categories  in the respective period, group (IG/CG), age, sex, BMI, smoking status, employed before PR (yes/no), and previous HRQL at T0, T1, or T2 respectively. ∆ACT = 0 was the reference group. The ACT categories are based on the approach of Sullivan et al. , who anaWe measured the GROC at T2 and T3 and considered a small GROC change as the minimal important change. We calculated the MID separately for improvement and deterioration, as well as combined using the absolute value of the changes. In analogy to Juniper et al. , who anaThe study sample included 371 patients: 199 (53.6%) were in the CG and 172 (46.4%) in the IG. The mean age was 51.4 years (SD: 5.6), and 58.5% of the population was male. Around 50% of the patients were current or previous smokers, and more than 80% were employed before the PR. Baseline HRQL did not differ in the groups, HRQL gains of the IG exceeded that of the CG regarding every measure. The whole development of the HRQL stratified by groups can be seen in Table None of the questionnaires used showed floor effects at any time point. Only the EQ-5D index showed ceiling effects at T2 and T3 with 55 (32%) patients each   and 0.78 CI ); VAS showed moderate ICC (0.62 CI [0.53: 0.70]).At T2, there were 185 (49.9%) patients in ACT-A, 72 (19.4%) in ACT-B, and 114 (30.7%) in ACT-C. At T3, there were 164 (44.2%) patients in ACT-A, 94 (25.3%) in ACT-B, and 113 (30.5%) in ACT-C. Adjusted mean scores for the ACT groups at T2 and T3 can be found in Fig. The overall responsiveness of a change in asthma control (measured in categories) of the HRQL tools was moderate. In most cases, AQLQ and VAS could differentiate between patients staying stable vs. patients reaching the |MID| on the ACT scale. The EQ-5D index was responsive to changes in only one period  Table ; howeverAccording to GROC at two time points, we identified (combining deterioration and improvement) mean MIDs in the pooled analysis of 0.67  for AQLQ, 12.28  for VAS, and 0.09  for the EQ-5D index  and different severity levels (ACT-A|ACT-B vs. ACT-B|ACT-C), but lower. Furthermore, AQLQ and VAS had a higher ability to differentiate between patients with asthma control or notand without asthma control (ACT-A vs. ACT-B) compared with differentiating between not well-controlled and very poorly controlled asthma (ACT-B vs. ACT-C). As the goal is to reach asthma control for most of the interventions, the differentiation between different degrees of uncontrolled not controlled asthma might be considered of secondary value. The results suggest that AQLQ, the EQ-5D index as well as VAS are all suited to detect patient groups with low HRQL and greater need for disease control, e.g., patients eligible for PR. Hernandez et al.  conducte and diffAn important aspect in health economics is the evaluation of health interventions. Therefore, HRQL tools should be reliable and responsive to changes to enable evidence-based recommendations regarding health care interventions. In a longitudinal approach, we assessed reliability (ICC) between T0 and T1, where none of the patients had yet received PR and their ACT score stayed stable. Reliability was moderate for VAS but good for the EQ-5D index and AQLQ. Without interventionA, asthma-related components of HRQL without intervention tend to be more stable than generic health, which might explain the observed higher reliability of the AQLQ. Additionally, AQLQ reflects a time period of 4 weeks, whereas EQ-5D-5 L asks for current health only, which increases the volatility of the measurements. Nevertheless, all instruments are suitable for repeated measurements.We assume that PR improves asthma control and clinical parameters and thus positively affects (at least disease-specific) HRQL. Therefore, in our pooled analysis, we had subgroups experiencing improvement (mostly in the IG) and patient groups staying relatively stable (mostly in the CG). This allowed us to examine HRQL changes in a heterogeneous study population. AQLQ was sensitive to big positive and negative changes (changes ≥|MID|). VAS was also able to differentiate between patients with deteriorating or improving HRQL by more than the MID-ACT, but not between small negative or positive changes. Given that the reference group for all HRQL tools is “no change”, a detection of changes below MID is very challenging because of the slight differences from the reference level. The EQ-5D index in our sample could not differentiate significantly between patients reaching a clinically relevant change on ACT (MID) or not, except for one case. This might be an issue regarding cost–utility studies using QALYs as the primary outcome, as suggested by the National Institute for Health and Care Excellence guidelines because, even if patients reach a clinically relevant increase in ACT (MID) through an intervention, it might be overlooked by the EQ-5D index. Thus, the intervention would not be considered cost effective. Looking at the quantile regression approach, a slightly different pattern emerged, where the EQ-5D index detects changes. However, we believe that the magnitude of the change on the EQ-5D index does not match the change on the ACT (e.g. at quantile 0.5 a MID change on ACT only changes the EQ-5D index by approximately 20% of its estimated MID), and leaves a significant improvement on the ACT undetected. Cost–utility studies should thus consider other secondary outcomes, which can potentially evaluate these changes. Similar results were reported from Sullivan et al. ; howeverUsing the GROC to identify the MID for the AQLQ resulted in a slightly higher MID than previous literature would suggest (0.65 vs. 0.5) . HoweverSzende et al.  used theAlthough the EQ-5D index showed slightly worse properties than the AQLQ, we should be aware of the different approaches behind the questionnaires. Generic questionnaires cover broad life aspects and facilitate comparisons among different disease groups, whereas disease-specific measures are for within-group comparisons. Furthermore, regarding the responsiveness of the tools to an ACT change is easier for the AQLQ, as it measures similar aspects and thus has overlapping content, whereas the EQ-5D index lacks asthma-specific content and can only indirectly measure such a construct , 38.There are some limitations to this study. As the EQ-5D assesses current health, whereas the AQLQ has a timeframe of 2 weeks and the ACT of 4 weeks, there is a potential bias while comparing these measures directly. Because asthma has a varying intensity, depending on the asthma attacks, valuing health on a single day may lead to distorted results.Additionally, there is a chance that HRQL tools behave differently in the control vs intervention CG vs. the IG, and a stratified analysis would be recommended. To achieve a sufficiently high n, we conducted a pooled analysis, but we think that our adjustment for the group variable best possibly accounted for this issue.The generalizability of the results is not necessarily given for patients outside Germany. Furthermore, patients with initially controlled asthma were not included in this analysis; therefore, we might miss important aspects about mild asthma cases. Nevertheless, the number of patients in this randomized controlled setting was high, and we believe our results are still valuable for the examined disease group.In conclusion, all presented HRQL tools had good discriminatory power and good reliability. However, EQ-5D-5L had difficulties in detecting  changes in disease control. Nevertheless, EQ-5D is still an important tool to compare HRQL across disease areas and to facilitate health economic evaluations, also in the field of asthma. Therefore to draw a more comprehensive picture, we would suggest using supplementary measures  to EQ-5D-5L to evaluate asthma-specific interventions. Nevertheless, it is still an important tool to compare HRQL across disease areas and to facilitate health economic evaluations.Additional file 1.


Radiation pneumonitis (RP) could be a lethal complication of lung cancer treatment. No reliable predictors of RP severity have been recognized. This prospective pilot study was performed to identify early predictors of high grade lung toxicity and to evaluate clinical, biological or dosimetric features associated with different grades of toxicity.18F-2-fluoro-2-deoxy-D-glucose positron-emission tomography was performed before radiotherapy (RT), after three weeks of treatment, and two months post-RT. For analysis, patients were grouped by grade (low [G1-G2] vs. high [G3-G5]). The two groups were compared to identify predictors of RP. Protein expression BAL and lung tissue metabolism was evaluated in two patients (RP-G1 vs. RP-G3). Categorical variables such as comorbidities, stages and locations were summarized as percentages. Radiation doses, pulmonary function values and time to RP were summarized by medians with ranges or as means with standard deviation. Longitudinal analysis PFT was performed by a T-test.Sixteen patients with non-small cell lung cancer with indication of concurrent chemoradiotherapy using 60 Gy/2 Gy/fraction starting at cycle one of platinum based chemotherapy were included. Bronchoalveolar lavage (BAL), pulmonary function testing (PFT), and p = 0.02) in diffusing lung capacity for carbon monoxide (DLCO) after three weeks of RT. No correlation between dosimetric values and RP grades was observed. BAL analysis of the selected patients showed that CXCL-1, CD154, IL-1ra, IL-23, MIF, PAI-1 and IFN-γ were overexpressed in the lungs of the RP-G3 patient, even before treatment. The pre-RT SUVmax value in the RP-G3 patient was non-significantly higher than in the patient with RP-G1.All 16 patients developed RP, as follows: G1 ; G2 ; G3 ; and G5 . Patients with high grade RP presented significant decrease (RT induces some degree of RP. Our data suggest that decrease in DLCO% is the most sensitive parameter for the early detection of RP. Moreover, we detect biological differences between the two grades of pneumonitis, highlighting the potential value of some cytokines as a prognostic marker for developing high grade lung toxicity. Further multicenter studies with larger sample size are essential to validate these findings. However, its effectiveness is limited by the risk of radiation-induced lung injury (RILI). RILI is the result of an abnormal healing response to lung irradiation caused by damage to parenchymal cells, vasculature, and/or stroma followed by inflammatory cytokine release ; however, V20 was lower in the high-grade group [29.5 Gy (95% CI 23–30) vs. 32 Gy (95% CI 21–35)]. No significant correlation between dosimetric values and RP grades was observed.Table p = 0.0203)   and DLCO(%) at diagnosis was not associated with the different grade of RP development. No significant differences in mean values were found between the groups at the three time points  Tables  and 4. HDifferent cytokine and chemokine expression profile (pre-RT and week 3) was found in a patient with RP-G1 compared to another with RP-G3 Fig. . Before The pre-RT SUVmax value was calculated for the RP-G1 and RP-G3 patients. In both patients, the pre-treatment SUVmax was higher than normal  . HoweverThe present study demonstrates that RT induces RILI in all patients who undergo external RT with variable clinical manifestations and different degrees of lung damage. This variability in the degree of RILI suggests that severe RP may be associated with differences in individual predisposition. The early decrease of DLCO(%) was a predicting factor of severe RP development and thus could serve as a marker for early diagnosis and treatment modification.The reported prevalence of RP in lung cancer patients ranges from 0 to 58% . This vaPrevious studies have shown that subtle, local changes in PFT after RT can be used as indicators of acute and chronic lung damage, although published results are not always consistent , 9. The Classically, RP grade has been associated with the radiation dose , 8. In o18FDG-PET/CT has been use to quantify increase cell glycolysis in the healthy lung tissue, excluding tumoral areas, like a marker of pulmonary inflammation through the uptake of 18FDG mesure by SUV [The e by SUV -17. It se by SUV . This fie by SUV .18F-FDG lung uptake in the subsequent development of RP symptoms. In this study, RP-G3 patient had a higher SUVmax in healthy lung tissue respect RP-G1 patient.These findings support the experience already published by Catillo et al.Accordingly, Castillo et al. , 17 demoRILI produce an imbalance between type 1 and type 2 helper T-cells and abnormal fibroproliferative wound healing . VariatiWe have to recognize some limitations: First, the small sample size which was calculated using “observed versus a reference mean” and even although we have enrolled sixteen patients, instead of including seventeen, we have not had any loss of follow up. Secondly, the low power of the biological lung and the PET/CT image analysis (only two patients). In this sense, this is a pilot study to identify if there are differences in biological features associated with different grades of RP. Our findings, warrant further investigation in a larger sample. The main strength of the study is that it is the first prospective study to evaluate patients with NSCLC through a longitudinal clinical and biological follow-up that demonstrate RT induces RILI in all cases but in some of them with a high-grade of lung injury and consequent altered wound repair.RT treatment always induces some degree of lung injury and the extent of the damage is variable. Our data suggest that decrease in DLCO% is the most sensitive parameter for the early detection of severe RP. Moreover, we detect biological differences between the two grades of pneumonitis, highlighting the potential value of cytokines such as CXCL-1, CD154, IL-1ra, IL-23, MIF, PAI-1 and IFN-γ as a prognostic marker for developing high grade of lung toxicity. Further multicenter studies with larger sample size are essential to validate these preliminary findings.